The complex process of protein translocation across cell membranes is a common theme in bacterial pathogenesis. A particularly efficient molecular machine for pro tein delivery is the type III secretion (T3S) system. The T3S system acts as a syringe that injects proteins from the bacterial cytoplasm directly into the cytoplasm of a target cell. Once into the host cytosol, translocated toxins subvert eukaryotic cellular processes (e.g., blocking phagocytosis), modulating the host response in favor of infection. Collected evidence in Yersinia pestis and Pseudomonas aeruginosa suggests that two secreted T3S proteins insert into the target cell membrane and form a pore (or translocon) through which toxins are translocated. Despite recent advances on the char- acterization of these two proteins (the translocators), their structure and mechanism of assembly of the translocon remain unknown. I have developed a set of fluorescence techniques that have been successfully used to characterize the structure and pore-formation mechanism of various homo- oligomeric cytolytic toxins. I now propose to extent the use of these techniques to multi- protein transmembrane complexes, like the T3S translocon. The fluorescence approach will be combined with other biochemical and biophysical techniques (e.g., electrophysi- ology measurements, single molecule techniques, and cryo-electron microscopy) to un- ambiguously address fundamental structural aspects of the T3S translocon structure and assembly. By selective incorporation of various probes (e.g., environment-sensitive fluorophores, crosslinkers, gold-nanoparticles, charged groups, etc.) in the P. aerugi- nosa translocators, we will experimentally identify, among other things: which segments of these proteins are essential to determine the characteristics of the translocon channel, what segments form the contact interface between the needle and the translocon, and how the translocators are arranged in the translocon complex formed in the mammalian cell membrane. In addition to elucidate fundamental aspects of translocon assembly into lipid bi- layers, these studies may ultimately lead to novel therapeutic strategies that block pro- tein translocation and interfere with bacterial colonization in a broad variety of threaten- ing human pathogens.